PROJECT SUMMARY Hearing impairment is one of the most common sensory disabilities, 250 million people worldwide have moderate to severe hearing loss (38), and significantly reduces quality of life due to the central role of verbal communication. On an economic scale, the total negative impact of hearing loss is greater than that of multiple sclerosis, spinal cord injury, stroke, epilepsy, Parkinson's and Huntington's disease combined and effects 4 times as many people (68). The most common causative factor among the defined hearing loss etiologies is excessive noise, and millions of people are exposed to dangerously loud noise at work. We hope that our research findings will aid in the reduction of noise-induced hearing loss by identifying significant proteins and pathways responsible for hearing loss. Specifically, we have developed a quantitative proteomic analysis platform to probe the effect of excess noise on the cochlear proteome. Our preliminary data shows this approach can accurately measure thousands of proteins from a single mouse and has already revealed proteins significantly perturbed after noise exposure. We have also made progress isolating the organ of Corti to ensure the accurate measurement of low abundant but potentially altered proteins in hair and adjacent support cells, and in cochlear nerve synapses. More specifically, we will quantitatively analyze inner ear extracts from mice exposed to multiple levels of noise. Through these comparative exposures, we will differentiate proteins with characteristics that are impacted by excess noise. The candidates from these proteomic experiments will be explored with bioinformatic tools and validated by traditional antibody based approaches. Next we will develop biochemical methods to ensure the accurate measurement of rare low abundance proteins. We will also test if protein?protein interactions are disrupted without significant changes in expression levels. Finally we will use bioactive molecules known to protect from NIHL and repeat the proteomic analysis to investigate the mechanisms by which these drugs are effective. In particular, we think that a comprehensive understanding of the inner ear proteome will accelerate the greater research field of hearing injury. In summary, we propose here to identify and investigate molecular defects in NIHL by applying quantitative proteomic tools that can simultaneously and sensitively investigate thousands of proteins in a single analysis. We believe this proposal represents the first ever application of quantitative proteomics to the investigation of NIHL and may hold the required analytical strength to kick start the development towards effective therapeutics to eventually treat and prevent NIHL.